In the last few years, rapid accumulation of genome sequences and protein structures has been paralleled by major advances in sequence database search methods. The powerful Position-Specific Iterating BLAST (PSI-BLAST) method developed at the NCBI formed the basis of our work on protein motif analysis. During last year, we made further progress in detailed analysis of the classification, evolution, and functions of several classes of proteins. In particular, a new major class of P-loop NTPases was described, the so-called STAND class, after signal transduction ATPases with numerous domains. The STAND class includes the AP-ATPases (animal apoptosis regulators CED4/Apaf-1, plant disease resistance proteins, and bacterial AfsR-like transcription regulators) and NACHT NTPases (e.g. NAIP, TLP1, Het-E-1) that have been studied extensively in the context of apoptosis, pathogen response in animals and plants, and transcriptional regulation in bacteria. We show that, in addition to these well-characterized protein families, the STAND class includes several other groups of (predicted) NTPase domains from diverse signaling and transcription regulatory proteins from bacteria and eukaryotes, and three Archaea-specific families. We identified the STAND domain in several biologically well-characterized proteins that have not been suspected to have NTPase activity, including soluble adenylyl cyclases, nephrocystin 3 (implicated in polycystic kidney disease), and Rolling pebble (a regulator of muscle development); these findings are expected to facilitate elucidation of the functions of these proteins. The STAND class belongs to the additional strand, catalytic E division of P-loop NTPases together with the AAA+ ATPases, RecA/helicase-related ATPases, ABC-ATPases, and VirD4/PilT-like ATPases. The STAND proteins are distinguished from other P-loop NTPases by the presence of unique sequence motifs associated with the N-terminal helix and the core strand-4, as well as a C-terminal helical bundle that is fused to the NTPase domain. This helical module contains a signature GxP motif in the loop between the two distal helices. With the exception of the archaeal families, almost all STAND NTPases are multidomain proteins containing three or more domains. In addition to the NTPase domain, these proteins typically contain DNA-binding or protein-binding domains, superstructure-forming repeats, such as WD40 and TPR, and enzymatic domains involved in signal transduction, including adenylate cyclases and kinases. By analogy to the AAA+ ATPases, it can be predicted that STAND NTPases use the C-terminal helical bundle as a "lever" to transmit the conformational changes brought about by NTP hydrolysis to effector domains. STAND NTPases represent a novel paradigm in signal transduction, whereby adaptor, regulatory switch, scaffolding, and, in some cases, signal-generating moieties are combined into a single polypeptide. The STAND class consists of 14 distinct families, and the evolutionary history of most of these families is riddled with dramatic instances of lineage-specific expansion and apparent horizontal gene transfer. The STAND NTPases are most abundant in developmentally and organizationally complex prokaryotes and eukaryotes. Transfer of genes for STAND NTPases from bacteria to eukaryotes on several occasions might have played a significant role in the evolution of eukaryotic signaling systems. Additionally, we identified a previously uncharacterized family of P-loop NTPases, which includes the neuronal membrane protein and receptor tyrosine kinase substrate Kidins220/ARMS, which is conserved in animals, the F-plasmid PifA protein involved in phage T7 exclusion, and several uncharacterized bacterial proteins. We refer to these (predicted) NTPases as the KAP family, after Kidins220/ARMS and PifA. The KAP family NTPases are sporadically distributed across a wide phylogenetic range in bacteria but ammong the eukaryotes are represented only in animals. Many of the prokaryotic KAP NTPases are encoded in plasmids and tend to undergo disruption to form pseudogenes. A unique feature of all eukaryotic and certain bacterial KAP NTPases is the presence of two or four transmembrane helices inserted into the P-loop NTPase domain. These transmembrane helices anchor KAP NTPases in the membrane such that the P-loop domain is located on the intracellular side. We show that the KAP family belongs to the same major division of the P-loop NTPase fold with the AAA+, ABC, RecA-like, VirD4-like, PilT-like, and AP/NACHT-like NTPase classes. In addition to the KAP family, we identified another small family of predicted bacterial NTPases, with two transmembrane helices inserted into the P-loop domain. This family is not specifically related to the KAP NTPases, suggesting independent acquisition of the transmembrane helices. CONCLUSIONS: We predict that KAP family NTPases function principally in the NTP-dependent dynamics of protein complexes, especially those associated with the intracellular surface of cell membranes. Animal KAP NTPases, including Kidins220/ARMS, are likely to function as NTP-dependent regulators of the assembly of membrane-associated signaling complexes involved in neurite growth and development. One possible function of the prokaryotic KAP NTPases might be in the exclusion of selfish replicons, such as viruses, from the host cells. Phylogenetic analysis and phyletic patterns suggest that the common ancestor of the animals acquired a KAP NTPase via lateral transfer from bacteria. However, an earlier transfer into eukaryotes followed by multiple losses in several eukaryotic lineages cannot be ruled out.